isopropyl-thiogalactoside and cyanopindolol

The relationship between rat beta(1)- and beta(2)-adrenergic receptors (ARs) and cyclic AMP (cAMP) responses was examined by inducible expression of each subtype in transfected GH(3) pituitary cells. Increasing expression of beta(1)- or beta(2)-ARs in stably transfected subclones increased basal cAMP, increased the potency of isoproterenol in stimulating cAMP formation, but did not change the maximal response. A linear relationship was observed between log B(max) and -log EC(50) for isoproterenol, with no significant differences between beta(1)- and beta(2)-ARs. When both subtypes were coexpressed at different densities and ratios, pharmacological analysis showed that both selective and nonselective agonists exerted their effects at least partially through both subtypes. Either subtype alone activated a maximal response when the other subtype was blocked, indicating a complete redundancy in coupling. Agonists could activate responses through either subtype, with responses mediated primarily through the subtype where the agonist was most potent. The nonselective agonist isoproterenol had similar potencies for activating both subtypes; however, the density and ratio of subtypes affected the relative potencies of the selective agonists norepinephrine and zinterol. We conclude that beta(1)- and beta(2)-ARs have similar coupling efficiencies in GH(3) cells, these efficiencies are not altered by coexpression of another subtype, they couple redundantly to cAMP formation, and the relative densities of beta(1)- and beta(2)-ARs control the potencies of selective agonists.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Binding, Competitive; Cells, Cultured; Cyclic AMP; Imidazoles; Iodine Radioisotopes; Isopropyl Thiogalactoside; Isoproterenol; Kinetics; Pindolol; Pituitary Gland, Anterior; Propanolamines; Rats; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Transfection

We examined the role of beta 1- and beta 2-adrenergic receptor (AR) density and ratio in catecholamine-stimulated cAMP responses in rat C6 glioma cells. These cells, which normally express both subtypes, were stably transfected with an isopropylthio-beta-D-galactoside-inducible vector containing either beta 1AR or beta 2AR coding sequences, and receptor expression was controlled by the time and concentration of isopropylthio-beta-D-galactoside exposure. Induction of the dominant beta 1AR subtype increased the potencies of isoproterenol (ISO) and other agonists in stimulating cAMP accumulation by 20-40-fold without changing maximal response. Induction of beta 2AR expression caused 7-13-fold increases in the potency of ISO, epinephrine, and zinterol, but not of norepinephrine, and a 20-40% loss in maximal response to all agonists. Selective antagonists showed that both subtypes contributed in a nonadditive manner in the response to ISO under different conditions. After beta 2AR induction, the effects of ISO were not blocked by the beta 1-selective antagonist CGP 20712A but were shifted 100-fold to the right by the beta 2-selective antagonist ICI 118,551. However, in the presence of ICI 118,551, CGP 20712A caused an additional 100-fold decrease in ISO potency, and Schild analysis revealed complex interactions between the two subtypes. Each antagonist alone caused smaller shifts to the right in the dose-response curve to NE and, when present simultaneously, completely abolished the NE response. We conclude that beta 1ARs and beta 2ARs have different efficiencies in activating cAMP accumulation in C6 glioma cells. Activation of coexisting subtypes results in complex and sometimes synergistic interactions between the two subtypes, which vary with agonist concentration, selectivity, subtype density, and ratio.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Colforsin; Cyclic AMP; Epinephrine; Gene Expression Regulation, Neoplastic; Genomic Library; Glioma; Imidazoles; Isopropyl Thiogalactoside; Isoproterenol; Kinetics; Norepinephrine; Pindolol; Radioligand Assay; Rats; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Recombinant Proteins; Transfection; Tumor Cells, Cultured